Paraffin has been used for many years as an embedding medium in the preparation of tissue specimens for sectioning in a microtome to produce specimen sections for histological studies. Such embedding processes generally include the well known steps of specimen fixation, dehydration, clearing, paraffin infiltration or impregnation, blocking or embedding in a block of paraffin, slicing the block and specimen into thin sections, mounting the sections on slides, removing the paraffin and solvents employed for this purpose (deparaffinizing), and staining the sections prior to microscopic analysis. The primary purpose of the embedding medium is to permit the specimens to be sectioned and mounted in the natural state. Plastic resins have also been used as embedding medium to provide a harder specimen that allows cutting of thinner sections. However, the use of paraffin-embedding has the advantage that the wax can be dissolved away from specimens prior to staining, allowing sections to be stained in the form of naked slabs of biopolymer and avoiding the extra difficulties and artifacts associated with the presence of unremovable resin-embedding medium (Horobin 1991).
Recent improvements in paraffin-embedding compositions broaden its applicability while maintaining its compatibility with downstream manipulation and analysis of samples. For example, an improved paraffin-based embedding material, which includes a mixture of paraffin and an effective amount of ethylene-vinyl acetate copolymer (0.5% to 5% by weight of paraffin) allows shorter infiltration time and thinner sections (U.S. Pat. No. 4,497,792). Another improvement, the double-embedding technique, yields sections of thin tissue membranes, such as rodent mesenteric membranes that usually measure only 10 microns in thickness. In this method several membranes are fixed and mounted on four needles located at the bottom of a plastic box and then embedded in agarose. The agarose block is removed, dehydrated in alcohol, cleared with HistoPetrol (tradename for a mixture of isoparaffin hydrocarbons), permeated with paraffin and sectioned. The observed tissue morphology is comparable to that obtained with methacrylate plastic embedding but is less time-consuming, less hazardous since no plastic hardener and activator are used, and makes immunohistochemical studies easier (Ghassemifar et al. 1992).
Consequently, deparaffinization of fixed, e.g. formalin fixed, paraffin embedded tissue sections is still a widely used methodology, particularly in hospital histopathology laboratories for immunodiagnostic purposes.
Xylene, which is a flammable, volatile and toxic organic solvent, is currently commonly used in protocols to solubilize paraffin for deparaffinization of specimen sections. Typically, the microscope slide-mounted specimen is immersed in a xylene bath until the paraffin is solubilized. The deparaffinized specimen is then washed with a series of alcohol solutions of decreasing alcohol concentration, typically as baths in which the specimen is immersed, to remove xylene before a final wash with water. Efforts have been made to replace xylene in the deparaffinization process with less toxic and less volatile solvents. Terpene oil (e.g. available under the tradename AmeriClear from Baxter Health Care Diagnostics, Inc. McGaw Park, Ill.) and isoparaffinic hydrocarbons (e.g. available under the tradename Micro-Clear from Micron Diagnostics, Inc., Fairfax, Va.) produced equal deparaffinization compared to xylene (Jones et al. 1993). However, a series of alcohol washes were still required to remove either solvent prior to the water wash to achieve compatibility with most types of staining, particularly immunohistochemical staining. Furthermore, the use of paraffin-embedded specimens with automated systems, such as immunostainers, is increasing.
Accordingly, there is still a need for deparaffinization compositions and methods that can effectively remove paraffin or improved paraffin-based embedding materials from specimens prior to histochemical or other diagnostic analyses, while minimizing danger to users, allowing compatibility with automated systems, and maintaining compatibility with downstream analyses. Deparaffinization compositions and methods that entail no or limited toxicity or carcinogenicity, produce no or minimal odors, reduce the quantity of toxic solvents used, minimize hazardous wastes, and/or decrease corrosiveness and flammability are needed.
Cited Literature
1. Horobin, R. W., In Histochemical and Immunochemical Techniques: Application to pharmacology and toxicology, (1991) Bach, P. and Baker, J., eds., Chapman & Hall, New York, N.Y. pp 1-9.
2. Ghassemifar, R. et al. (1992) “A double-embedding technique for thin tissue membranes” Biotech. Histochem. 67:363-366.
3. Jones, R. T. et al. (1993) “Comparison of deparaffinization agents for an automated immunostainer” J. Histotechnology 16:367-369.
4. Mullin, L. S. et al. (1990) “Toxicology update isoparaffinic hydrocarbons: a summary of physical properties, toxicity studies and human exposure data” J. Appl. Toxicol. 10:135-142.